Electrophoresis is a scientific technique of moving charged particles, using an electric field, through solid or semi-solid media. The technique is commonly used in scientific research, including medical laboratories for analyzing various blood proteins.
In the analysis of biological material, it is known that much information can be provided by identification and qualification of certain components, such as proteins or nucleic acid fragments. It is well known that electrophoresis is an effective method of identifying the respective components of such biological material for subsequent microscopic analysis of the sample or for subsequently employing optical densitometry techniques in analyzing the samples.
In the basic method of electrophoresis, an electric field is applied across a diffusion medium. The diffusion medium contains biological particles having an electric charge. As the electric field is applied, the biological particles migrate through the diffusion medium at different rates, depending on the amount of charge on the particles and size of the particles. Thus, the biological particles are separated into distinct bands as a result of the differential migration. Subsequent staining of the fractional components in the diffusion medium allow visual analysis by optical densitometry or other methods.
Electrophoresis has been performed through a series of manual steps for many years. The manual process typically starts with preparing an electrophoresis chamber. The chamber is used to create the electric field. The chamber includes a rectangular electrically insulated container having electrodes at opposite ends thereof. The chamber is then filled with a buffer to create the interface between the two electrodes for supporting the electric field. The buffer is typically a stable aqueous solution. Next, the operator must prepare the diffusion medium, such as SDS, cellulose acetate or agarose gel. The gel is set upon a support medium, such as MYLAR™, so as to maintain the structural integrity of the gel during handling.
After the operator has prepared the electrophoresis chamber, the operator then manually applies, as precisely as possible, consistent volumes of the biological samples to precise locations in the diffusion medium or gel. The operator then places the gel into the electrophoresis chamber so that the edges of the gel are aligned with electrodes at each of the longitudinal ends of the chamber. The electric field is applied using a precise and consistent high voltage applied for a precise and consistent interval of time across the gel in the buffer solution.
After the electric field has been removed, the operator manually moves the gel into another container to apply a uniform coating of staining reagent to the surface of the gel, thus allowing a precise and consistent interval of time for the reagent and sample to chemically react. The staining reagent can be a colormetric or enzymatic solution, used to chemically combine with the bands of the biological particles of the sample, thereby causing the bands to exhibit optical characteristics. The operator may place the gel into a temperature controlled oven and incubate the gel, using a precise and consistent temperature and time interval. Incubation controls the chemical reaction between the bands and the staining reagent by means of applying heat for a fixed interval of time. The operator then dries the gel by increasing the oven temperature for a second precise and consistent temperature and time interval. The drying process stops the reaction between the bands and the reagent.
There have been prior art apparatus and methods available for automatically performing electrophoresis and staining of the plurality of samples applied to a diffusion medium. For example, U.S. Pat. No. 4,360,418 to Golias and U.S. Pat. No. 4,391,689 to Golias describe an automated electrophoresis and staining apparatus and method. Such prior art includes an electrophoresis chamber and a series of vats mounted upon a platform and arranged in a row where the vats are adapted to contain, respectively, a liquid stain and a series of plate processing solutions. The plate holder rack, having a horizontal open frame, supports an upright electrophoresis plate or support medium onto which has been applied a sample for electrophoretic fractionization. Such an electrophoresis plate had to have been previously prepared by applying samples either manually or by using one of the parallel applicators described above. The plate is nested within the chamber within an electrophoretic circuit for a predetermined time period. A power-operated lift and transfer assembly is provided on the base and is adapted to lift, transfer and lower the plate holder rack and plate from the chamber progressively into each of the underlying vats for a predetermined period of time in a linear stepping motion maintaining the plate in an upright position at all times. It is noted that the staining process relies on chemical procedures for the staining process rather than the manual system described above where incubation and drying are used.
U.S. Pat. No. 4,890,247, issued Dec. 16, 1989 to Sarrine et al., and owned by the present applicant, describes an automatic electrophoresis apparatus and method. In this patent, an electrophoresis machine is described in which all electrophoresis processing, scanning and densitometer functions are automatically performed under computer control. This machine includes an apparatus for automatically pipetting samples from the sample plate to the surface of the support strip. There is also an apparatus for applying electric current to the strip while simultaneously cooling it. Additionally, and furthermore, the device includes apparatus for applying and spreading fluorescent staining reagents to the strip. The device includes a special apparatus for incubating the strip and subsequently drying the strip. A video camera, mounted to a fluorescent scanning box which encloses the application plate, is used in combination with digital processing equipment so as to electronically scan the electrophoretically longitudinally displaced components of the samples for determining relative component densities. The subject matter of this patent is also described in U.S. Pat. Nos. 4,810,348, 4,909,920, and 4,986,891. Each of these patents is also owned by the same assignee.
U.S. Pat. No. 5,460,709, issued on Oct. 24, 1995 to Sarrine et al., also discloses another type of automatic electrophoresis method and apparatus. This apparatus is used for automatically performing medical assays and includes an electrophoresis platform which cooperates with a gantry assembly. The electrophoresis platform and the gantry assembly are movable along paths that are perpendicular to each other. An applicator assembly includes pipettes which transfer samples in a specimen tray to a electrophoresis plate mounted on the electrophoresis platform. The electrophoresis platform then moves to a position into the gantry assembly, where electrophoresis is conducted to separate the samples into different fractions. The electrophoresis platform then moves between a reagent pouring station where a reagent is applied to make the separated fractions fluoresce under ultraviolet light. The electrophoresis platform is then moved beneath the gantry assembly again as an air knife in the gantry assembly spreads the reagent. After incubation and drying of the electrophoresis plate, the electrophoresis platform and the gantry assembly are moved relative to one another while the electrophoresis plate is read with the aid of ultraviolet lamps and a photomultiplier tube mounted in the gantry assembly. The gain of the photomultiplier tube is automatically adjusted and the data gathered is automatically edited to remove background noise.
It is object of the present invention to provide an improved method and apparatus for automatically conducting electrophoresis.
It is another object of the present invention to provide an electrophoresis method whereby a single pipette can be used for the various operations associated with the gel plate and with the staining of the electrophoresed plate.
It is another object of the present invention to provide an automatic electrophoresis method and apparatus whereby all the operations are carried out in situ.
It is another object of the present invention to provide an automatic electrophoresis method and apparatus in which the de-staining operation can be carried out in a convenient and easy manner.
It is a further object of the present invention to provide an automatic electrophoresis method and apparatus which allows a plurality of reagents to be dispensed by a single pipette so that various analyses can be carried out.
It is a further object of the present invention to provide an automatic electrophoresis method and apparatus which provides a single apparatus for automatically applying a plurality of biological samples to a diffusion medium, automatically subjecting such samples to electrophoresis, automatically staining such samples, automatically incubating and drying the support medium in which the components of the samples have been separated into longitudinal bands, and automatically scanning such bands so as to automatically display results.
These and other objects and advantages of the present invention will become apparent from a reading of the attached specification and appended claims.